GRANT=6546082;P01AG The realization that variant Creutzfeldt-Jakob disease (vCJD) in humans is acquired from bovine-spongiform encephalopathy (BSE)-contaminated food products has raised serious concerns regarding other routes of infection, including exposure to bovine extraction products in vaccines, and therapeutic blood transfusion from donors incubating or suffering from vCJD. Recent experimental transmission of BSE in rodents, sheep, and primate models through blood transfusion further underscores the risk of similar transmission in humans through blood components. To develop strategies that will prevent further spread of BSE and vCJD, it is necessary to identify pathway(s) by which the infectious agent is transported from peripheral sites to the brain. The principal infectious agent of BSE, vCJD, and other prion disorders is a conformationally transformed protein, the scrapie prion protein (PrP-Sc), which is responsible for both transmission and pathogenesis of these disorders. In this proposal, we will investigate the mechanism(s) by which orally ingested PrP-Sc is transported across the intestinal epithelial barrier, and across the blood brain barrier (BBB) to the brain parenchyma. Possible avenues include indirect spread via peripheral nerves, or directly from peripheral circulation to the brain parenchyma through the blood brain barrier (BBB). Since both intestinal epithelium an BBB are comprised of an impermeable layer of polarized cells with tight junctions, PrP-Sc may traverse these barriers using similar mechanisms. Thus, we will evaluate the cellular pathways by which PrP-Sc, a protein of approximately 27-30kDa, is transported across a monolayer of epithelial cells of the intestine, and endothelial cells of the BBB. Initial investigations will be carried out in vitro in cell models, and later confirmed in vivo in transgenic mouse models. Three Specific Aims will be addressed in this study: 1) The transport of a short peptide of PrP(106-126)(PrP[106-126]), a 7kDa of human PrP-Sc, and mouse PrP-Sc through a monolayer of intestinal (Caco-2 and HT-29) and endothelial (EC219) cell lines will be evaluated, representing in vitro models of human intestinal epithelium and blood brain barrier (BBB) respectively. 2) Similar investigations will be carried out using isolated segments of mouse intestine and cerebral microvessels in vitro. These models mimic the in vivo milieu more closely than cell models. 3) The transport pathway(s) of PrP[106-126] and PrP-Sc identified in Aims 1 and 2 will be confirmed in transgenic mouse models that lack (PrP[-/-]), or overexpress PrP (PrP[+/+]). Particular emphasis will be placed on accumulation of PrP-Sc in the spleen, and transport across the BBB in association with plasma protein(s) or hematopoietic cell(s), or independently. These investigations will provide possible avenues to intercept the passage of PrP-Sc to the brain parenchyma after accidental peripheral exposure to prion-infected material.